The hormone insulin is recognized as having actions that affect the trans-membrane transport of different substances, particularly glucose, into numerous different kinds of cells of the human body.
Insulin is a large polyteptide molecule with a molecular weight of 5808. It consists of a so-called A Chain and a so-called B Chain, connected together by two disulfide bridges. The hormone insulin is produced in the beta cells of the pancreas, and the stimulus for its secretion into the bloodstream is a function of an increase in blood glucose concentration.
Its action on the liver, adipose tissue, and skeletal muscle have all been studied in the literature in great detail, and it is now recognized that insulin also affects a wide variety of tissues in addition to these.
Apart from its membrane transport of glucose, insulin also regulates transport of some amino acids, certain fatty acids, the minerals potassium and magnesium, and certain monosaccharides. Further, it performs a mediation function by regulating the formation of macromolecules which are used in cell structure, cell energy storage, and the regulation of many cell functions. More particularly, it is known that glucose stimulates glycogenolysis, lipogenesis, proteogenesis, and nucleic acid synthesis. It also increase glucose oxidation and magnesium-activated sodium-potassium ATPase activity.
It is further known that there is a single mechanism involved in the initiation of all of the above biological affects and, particularly, this mechanism is the interaction of the hormone insulin with its specific cell receptor. The insulin receptor consists of two alpha subunits, each of molecular weight 135,000 and two beta subunits, each having a molecular weight of 95,000, which are linked together by disulfide bonds. The alpha unit is predominantly located upon the outer surface of the cell membrane, and the insulin binding/linkage domain is located there. The transmembrane beta unit contains tyrosine kinase activity on its cytoplasmic domain that results in rapid receptor autophosphorylation, that is, effective absorption of the beta subunit into the cell. Activation of the kinase toward exogenous substrates of the cell is, it appears, preceded by this insulin-dependent autophosphorylation reaction of the beta subunit. Action on other cellular substraites ultimately leads to the expression of the full range of insulin actions at the cellular level. See Schnetzler, Rubin, and Pilch. Structural Requirements for the Transmembrane Activation of the Insulin Receptor Kinase. J Biol Chem 261:15281-15287, 1986.
After insulin binds to the receptor with activation of the kinase, followed by receptor autophosphorylation, the insulin-receptor combination is endocytosed (absorbed) into the cell cytoplasm. This phenomena accounts for the down-regulation of insulin receptor activity within the blood that ensues following insulin stimulation. With this endocytosis, a variety of events may then take place. Insulin disassociates from the receptor and, following fusion of the endocytotic vesicle with cellular lysomes, it is degraded by lysomal enzymes. The free receptor may then itself be degraded by the lysomal enzymes, or it may recycle back to the surface (substrate) of the cell membrane. Finally, the free phosphorylated receptor may proceed to activate other substrates in the cytoplasm or may activate particular cellular organelles, e.g., the golgi apparatus and the nucleus, to produce the many cell changes referred to above. See Heidenreich and Olefsky. Metabolism of Insulin Receptors: Molecular Bases for Insulin Action. Page 163, Plenum Press, New York, 1985.
The most commonly recognized action of insulin is that of lowering blood glucose. This is accomplished via a process of facilitated diffusion across cell membranes. It has been hypothesized that the mechanism of this facilitated diffusion involves the translocation of a glucose transport protein from the cytoplasm out to the cell membrane (the exterior substrate). This translocation process involves the fusion of intracytoplasmic vesicles with the membrane of the cell. These vesicles contain the glucose transport protein in their enclosing membranes. Once exteriorized on the cell surface, the transport proteins of the vesicles serve as channels for glucose to enter the cell. This particular protein has been identified as a 40,000 molecular weight moiety that is associated with the golgi apparatus. See Burdett, Beeler and clip. Distribution of Glucose Transporters in Insulin Receptors in the Plasma Membrane and Transverse Tubules of Skeletal Muscle. Arch 8 Biochem Biopys 253:279-286, 1987.
The above process of translocation is reversible via endocytotsis of the membrane fragment containing said transport proteins, thus reconstituting the intra cytoplasmic vesicles. The whole activity of the glucose transport proteins is dependent upon metabolic energy and is independent of protein synthesis. See Kono, Translocation Hypothesis of Insulin Action on Glucose Transport. Federation Proc 43:2256-2257, 1984. The precise nature of the signal or messenger through which insulin turns this process on and off remains to be fully explained.
It is known that insulin receptors are widely distributed in mammalian organisms, there being in the range of 100 to 100,000 receptors per cell in different tissues. Rarely do any cells have no receptors at all. See Rosen. After Insulin Binds. Science 273:1452-1457, 1987.
A number of malignant neoplastic tissues have also been found to have a plentiful supply of insulin receptors, see Wong and Holdaway. Insulin Binding by Normal and Neoplastic: Tissue. Int J Cancer 35:335-341, 1985, perhaps reflecting cancer cell metabolism and the enhanced need that malignant cells have for glucose (see Cone, U.S. Pat. No. 4,935,450). Insulin may also play a role in the stimulation of cancer cell growth. See Myal, Shiu, Bhomic, and Bala. Receptor Binding and Growth Promoting Activity of Insulin-like Growth Factors and Human Breast Cancer Cells. Cancer Research 44:5486-5490, 1984.) A number of different cancers have been found to actually produce and secrete their own insulin. See Shamas, Dhurandhar, Blackar, Insulin-secreating Bronchial Carcinoid Tumor with Widespread Netastases. Am J Med 44:632-637,. And Pavelic, Popovic. Insulin and Glucogon Secretion by Renal Adeno-Carcinoma. Cancer 48:98-100, 1981.
Investigation of many of the actions of insulin upon insulin receptors in numerous species has demonstrated that the properties of insulin receptors in mammalian tissue are remarkably similar, irrespective of cell type. This being so, it may be anticipated that what the activated insulin/insulin-receptor complex does in one tissue, it will do in all. This would of course be dependent upon the existence of the necessary metabolic machinery within a particular tissue to react to insulin activation. It has been found that not all tissues are equally endowed in such regard. For example, the brain is a tissue which does not have insulin receptors, but which does have an insulin-dependent glucose transport mechanism. More particularly, insulin receptors are found both on the capillary endothelium of the blood brain barrier (BBB) as well as upon the glial elements within the substance of the brain. These receptors do not seem to play any role, in conjunction with insulin, in the transmembrane transport of glucose which is essential to proper brain metabolism. Rather, the capillary endothelium of the BBB has its own unique transport system for glucose, as well as for a number of other nutrient transport system substances such as choline, adenine, adenosine, lactate, glutamate, phenylalanine, and arginine. See Pardridge. Receptor-Mediated Peptide Transport Through the Blood-Brain Barrier. Endocrine Reviews 7:314-330, 1986.
The composition of this meager interstitial fluid of the brain is carefully controlled by the very selective functioning of the BBB. Having access to this space, the substances then have free access to the brain cells.
The glucose transport system in the brain responds to chronic changes in blood glucose levels. That is, the system is up-regulated during periods of hyperglycemia, and in like fashion is down-regulated during prolonged periods of hypoglycemia, such as can occur with poorly controlled diabetes. In the context of the instant invention, glucose transport across the BBB is insulin-independent, and yet insulin receptors are found on the same BBB capillary endothelium which carries the glucose transport system. This insulin transport system is just one of a number of peptide transport systems found on the BBB. Others carry the insulin-like growth factors I and II and transferrin. See Pardridge, supra. The BBB insulin receptor is a glycoprotein having structural characteristics typical of the insulin receptor in peripheral tissues. It may be part of a combined endocytosis and, exocytosis systems, that is, a transcytosis system for the transport of the peptide of focus through the BBB in humans. The transcytosis of insulin through the human BBB would, it appears, allow for distribution and circulation insulin into brain interstitial areas and insulin action upon brain cells. Through a non-receptor mechanism.
In skeletal muscle, insulin has been shown to deliver enzyme-insulin-albumin conjugates into the cell. This entire complex, it has been determined, is transported into the cell by a process resembling receptor-mediated endocytosis, and the enzyme-albumin-insulin complex is maintained by its enzymatic activity and its ability to bind antibodies to insulin. See Poznansky, Singh, Singh, and Fantus. Insulin: Carrier Potential for Enzyme and Drug Therapy. Science 223:1304-1306, 1984.
The instant invention reflects an elaboration of the above principles and, more specifically, those principals and methods set forth in our U.S. Pat. No. 4,971,951. In addition to said patent, other patented prior art has recognized the importance of the role of insulin as a carrier, adjuvant, or agent to enhance the absorption or to potentiate the effect of drugs administered to patients for the treatment of specific diseases. More particularly, U.S. Pat. No. 2,145,869, discloses a composition including insulin and glucose for the treatment of syphilis. Further, U.S. Pat. No. 4,196,196 (1980) to Tiholiz discloses a composition of insulin, glucose and magnesium dipotassium ethyline, diamine tetraacetic acid to enhance tissue perfusion and to facilitate a divalent-monovalent cation gradient. The general value and significance, in cancer treatment, of such a cation gradient, however facilitated, is recognized in U.S. Pat. No. 4,018,649 (1977) to Cone, entitled Process and Control of Cell Division.
A further U.S. Patent, namely, U.S. Pat. No. 4,277,465 (1981) to Kamada, teaches the use of an enamine derivative molecularly linked to insulin to facilitate its therapeutic absorption across the digestive tract.
The importance of insulin activity messengers is set forth in U.S. Pat. No. 4,839,466 (1989) to Saltiel.
The importance of insulin in the metabolism of malignant cells is, as noted above, recognized and discussed in U.S. Pat. No. 4,935,450 (1990) to Cone, entitled Cancer Therapy System for Effecting Oncolysis of Malignant Neoplasms.
Our above referenced U.S. Pat. No. 4,971,951 teaches a method of treatment of viral diseases including cancer and AIDS. The present invention is concerned with a broader method and means of systemic adjuvenation for potentiation of a broad range of therapeutic agents. In this sense, the instant invention may be viewed as an improvement of the invention of our said earlier U.S. patent.